Pathogen-induced macrophage apoptosis is an important mechanism used by several highly pathogenic bacteria to avoid detection by the innate immune system through killing of host macrophages, allowing them to establish highly virulent infections. We found that Bacillus anthracis, the causative agent of anthrax, can induce macrophage apoptosis and proposed that this process is an important contributor to its mechanism of pathogenecity. Using B. anthracis as a model, we found that induction of macrophage apoptosis requires inhibition of anti-apoptotic gene expression and activation of Toll-like receptor 4 (TLR4). Normally, TLR4 engagement results in macrophage activation and cytokine production, as well as induction of genes whose products prevent macrophage apoptosis, but B. anthracis uses its lethal toxin (LT) to inhibit activation of p38 MAP kinase (MAPK) and thereby prevents induction of macrophage survival genes. We identified several candidate macrophage survival genes whose induction is p38-dependent and is therefore LT-sensitive. We will continue with characterization of these genes and determination of their physiological role in the maintenance of macrophage survival. We also plan to identify the exact mechanism through which activation of p38 MAPK contributes to induction of these genes in response to TLR4 engagement. We have also identified the major mechanism through which TLR4 engagement can trigger macrophage apoptosis. The critical component of this mechanism is the double stranded (ds) RNA-responsive protein kinase PKR. Importantly, disruption of the gene encoding PKR protects macrophages from the apoptotic effect of B. anthracis, gersina and Salmonella. As these results were obtained ex vivo, we plan to study in detail the role of PKR in pathogen-induced macrophage apoptosis in vivo and determine whether inhibition of PKR can prevent macrophage apoptosis and reduce the burden and lethality of infections with macrophage-killing bacteria, such as B. anthracis. Assuming that PKR inhibition may provide a viable strategy for preventing pathogen-induced macrophage apoptosis, we will study in detail the mechanism through which TLR4 engagement leads to PKR activation. Interference with this activation process may be used to increase host resistance to bacterial infections, both with agents of bioterrorism and more common pathogens.